Temperature dependence of parasitic infection and gut bacterial communities in bumble bees.

High temperatures (e.g., fever) and gut microbiota can both influence host resistance to infection. However, effects of temperature-driven changes in gut microbiota on resistance to parasites remain unexplored. We examined the temperature dependence of infection and gut bacterial communities in bumble bees infected with the trypanosomatid parasite Crithidia bombi. Infection intensity decreased by over 80% between 21 and 37°C. Temperatures of peak infection were lower than predicted based on parasite growth in vitro, consistent with mismatches in thermal performance curves of hosts, parasites and gut symbionts. Gut bacterial community size and composition exhibited slight but significant, non-linear, and taxon-specific responses to temperature. Abundance of total gut bacteria and of Orbaceae, both negatively correlated with infection in previous studies, were positively correlated with infection here. Prevalence of the bee pathogen-containing family Enterobacteriaceae declined with temperature, suggesting that high temperature may confer protection against diverse gut pathogens. Our results indicate that resistance to infection reflects not only the temperature dependence of host and parasite performance, but also temperature-dependent activity of gut bacteria. The thermal ecology of gut parasite-symbiont interactions may be broadly relevant to infectious disease, both in ectothermic organisms that inhabit changing climates, and in endotherms that exhibit fever-based immunity.

Double-stranded RNA reduces growth rates of the gut parasite Crithidia mellificae.

Parasites of managed bees can disrupt the colony success of the host, but also influence local bee-parasite dynamics, which is regarded as a threat for wild bees. Therapeutic measures have been suggested to improve the health of managed bees, for instance, exploiting the bees' RNA interference (RNAi) pathway to treat against viral pathogens. Gut trypanosomes are an important group of bee parasites in at least two common managed bee species, i.e., managed Apis mellifera and reared Bombus terrestris. In several trypanosomes, RNAi activity is present, while in other associated genes of RNAi, such as Dicer-like (DCL) and Argonaute (AGO), it is lost. Up to date, the ability to exploit the RNAi of gut trypanosomes of bees has remained unexplored. Here, we screened parasite genomes of two honey bee protozoa (Crithidia mellificae and Lotmaria passim) and two bumble bee protozoa (Crithidia bombi and Crithidia expoeki) for the presence of DCL and AGO proteins. For C. mellificae, we constructed a double-stranded RNA (dsRNA) targeting kinetoplastid membrane protein-11 (KMP-11) to test the RNAi potential to kill this parasite. Transfection with KMP-11 dsRNA, but also adding it to the growth medium resulted in small growth reduction of the trypanosome C. mellificae, thereby showing the limited potential to apply dsRNA therapeutics to control trypanosome infection in managed honey bee species. Within bumble bees, there seems to be no application potentials against C. bombi, as we could only retrieve non-functional DCL- and AGO-related genes within the genome of this bumble bee parasite.

Large-scale cultivation of the bumblebee gut microbiota reveals an underestimated bacterial species diversity capable of pathogen inhibition.

A total of 1940 isolates from gut samples of 60 bumblebees representing Bombus pascuorum, Bombus terrestris, Bombus lucorum and Bombus lapidarius was collected and identified through state-of the-art taxonomic methods. The bacterial species diversity in these Bombus species exceeded that suggested by phylotype analysis through 16S rRNA amplicon sequencing, and revealed that B. pascuorum and B. terrestris had a unique microbiota composition, each. Representatives of most phylotypes reported earlier and detected in the present study were effectively isolated, and included several novel bacterial taxa and species reported for the first time in the bumblebee gut. Isolates were screened in pectin degradation assays and growth inhibition assays against the honeybee pathogens Paenibacillus larvae, Melissococcus plutonius and Ascosphaera apis and the bumblebee parasite Crithidia bombi. While inhibitory activity against each of these pathogens was observed, only one single culture was able to degrade pectin and polygalacturonic acid in vitro. The availability of accurately identified microbial isolates will facilitate future evaluation of the functional potential of the bumblebee gut microbiota.

Temperature-mediated inhibition of a bumblebee parasite by an intestinal symbiont.

Competition between organisms is often mediated by environmental factors, including temperature. In animal intestines, nonpathogenic symbionts compete physically and chemically against pathogens, with consequences for host infection. We used metabolic theory-based models to characterize differential responses to temperature of a bacterial symbiont and a co-occurring trypanosomatid parasite of bumblebees, which regulate body temperature during flight and incubation. We hypothesized that inhibition of parasites by bacterial symbionts would increase with temperature, due to symbionts having higher optimal growth temperatures than parasites. We found that a temperature increase over the range measured in bumblebee colonies would favour symbionts over parasites. As predicted by our hypothesis, symbionts reduced the optimal growth temperature for parasites, both in direct competition and when parasites were exposed to symbiont spent medium. Inhibitory effects of the symbiont increased with temperature, reflecting accelerated growth and acid production by symbionts. Our results indicate that high temperatures, whether due to host endothermy or environmental factors, can enhance the inhibitory effects of symbionts on parasites. Temperature-modulated manipulation of microbiota could be one explanation for fever- and heat-induced reductions of infection in animals, with consequences for diseases of medical and conservation concern.

A quantitative in vitro cultivation technique to determine cell number and growth rates in strains of Crithidia bombi (Trypanosomatidae), a parasite of bumblebees.

The protozoan parasite Crithidia bombi and its host, the bumblebee Bombus terrestris, are used as a model system for the study of the evolutionary ecology of host-parasite interactions. In order to study these interactions we established a method for in vitro cultivation of single parasite strains. Additionally, a high-throughput method is developed for the determination of cell numbers in cultures by means of optical density (OD) measurements. The protocol for in vitro cultivation allowed for growing different strains on agar plates as well as in culture medium. A calibration curve for the relationship between cell number and OD has been developed. Subsequently, growth rates for different genotypes of C. bombi have been recorded. Significant differences in the growth rates and generation times between these genotypes were demonstrated. As this might be related to the virulence of the parasite, this relationship may be confirmed by in vivo growth rate determination. In comparison with conventional cell counting, the application of OD measurements allows for high-throughput experiments as the time taken to record each sample is reduced by a factor of 30. The in vitro cultivation method allows for controlled infection experiments in order to study host-parasite interactions.

Bumblebee olfactory learning affected by task allocation but not by a trypanosome parasite.

Parasites can induce behavioural changes in their host organisms. Several parasite species are known to infect bumblebees, an important group of pollinators. Task allocation within bumblebee colonies can also cause differences in behaviour. Thus, task allocation may lead to context-dependent impacts of parasites on host behaviour. This study uses Bombus terrestris and its gut trypanosome Crithidia bombi, to investigate the effects of parasitism, task allocation (foraging or nest-work) and their interactions, on olfactory learning. Prior to undergoing the olfactory learning task, bees were orally infected with a field-realistic dose of C. bombi, and observed to determine task allocation. Parasitism did not significantly affect olfactory learning, but task allocation did, with foragers being significantly more likely to learn than nest bees. There was no significant interaction between parasitism and task. These results suggest that C. bombi is unlikely to affect pollination services via changes in olfactory learning of its host if bees are under no environmental or nutritional stress. However, wild and commercial colonies are likely to face such stressors. Future studies in the field are needed to extrapolate our results to real world effects.

Medicinal value of sunflower pollen against bee pathogens.

Global declines in pollinators, including bees, can have major consequences for ecosystem services. Bees are dominant pollinators, making it imperative to mitigate declines. Pathogens are strongly implicated in the decline of native and honey bees. Diet affects bee immune responses, suggesting the potential for floral resources to provide natural resistance to pathogens. We discovered that sunflower (Helianthus annuus) pollen dramatically and consistently reduced a protozoan pathogen (Crithidia bombi) infection in bumble bees (Bombus impatiens) and also reduced a microsporidian pathogen (Nosema ceranae) of the European honey bee (Apis mellifera), indicating the potential for broad anti-parasitic effects. In a field survey, bumble bees from farms with more sunflower area had lower Crithidia infection rates. Given consistent effects of sunflower in reducing pathogens, planting sunflower in agroecosystems and native habitat may provide a simple solution to reduce disease and improve the health of economically and ecologically important pollinators.

Phosphatidylcholine synthesis in Crithidia deanei: the influence of the endosymbiont.

In this study, the role of phospholipid biosynthetic pathways was investigated in the establishment of the mutualistic relationship between the trypanosomatid protozoan Crithidia deanei and its symbiotic bacterium. Although the endosymbiont displays two unit membranes, it lacks a typical Gram-negative cell wall. As in other intracellular bacteria, phosphatidylcholine is a major component of the symbiont envelope. Here, it was shown that symbiont-bearing C. deanei incorporates more than two-fold (32)Pi into phospholipids as compared with the aposymbiotic strain. The major phospholipid synthesized by both strains was phosphatidylcholine, followed by phosphatidylethanolamine and phosphatidylinositol. Cellular fractioning indicated that (32)Pi-phosphatidylcholine is the major phospholipid component of the isolated symbionts, as well as of mitochondria. Although the data indicated that isolated symbionts synthesized phospholipids independently of the trypanosomatid host, a key finding was that the isolated bacteria synthesized mostly phosphatidylethanolamine, rather than phosphatidylcholine. These results indicate that phosphatidylcholine production by the symbiont depends on metabolic exchanges with the host protozoan. Insight about the mechanisms underlying lipid biosynthesis in symbiont-bearing C. deanei might help to understand how the prokaryote/trypanosomatid relation has evolved in the establishment of symbiosis.

Serial passage of the parasite Crithidia bombi within a colony of its host, Bombus terrestris, reduces success in unrelated hosts.

In the wild, Bombus spp. bees may contract infections of the trypanosome parasite Crithidia bombi from their nestmates or from others while foraging on contaminated flowers. We expected that as C. bombi is transmitted repeatedly among related workers within a colony, the parasite population would become more successful in this relatively homogeneous host population and less successful in individuals from unrelated colonies of the same or different species. To test our prediction, we serially passaged cocktails of C. bombi strains through workers from the same colony, taking the intensity of infection in related versus unrelated workers as a measure of parasite success at each step in the serial transfer. Using a repeated measures ANOVA, we found the ability of C. bombi to exploit Bombus spp. hosts did not increase within a colony, but did decrease for infections in workers from unrelated colonies. This reduction in success is most likely due to a gradual loss of appropriate C. bombi strains from the infecting the population as the cocktail is 'filtered' during the serial passage within a given colony, without a corresponding increase in overall intensity of the surviving strains.

Insect antimicrobial peptides act synergistically to inhibit a trypanosome parasite.

The innate immune system provides protection from infection by producing essential effector molecules, such as antimicrobial peptides (AMPs) that possess broad-spectrum activity. This is also the case for bumblebees, Bombus terrestris, when infected by the trypanosome, Crithidia bombi Furthermore, the expressed mixture of AMPs varies with host genetic background and infecting parasite strain (genotype). Here, we used the fact that clones of C. bombi can be cultivated and kept as strains in medium to test the effect of various combinations of AMPs on the growth rate of the parasite. In particular, we used pairwise combinations and a range of physiological concentrations of three AMPs, namely Abaecin, Defensin and Hymenoptaecin, synthetized from the respective genomic sequences. We found that these AMPs indeed suppress the growth of eight different strains of C. bombi, and that combinations of AMPs were typically more effective than the use of a single AMP alone. Furthermore, the most effective combinations were rarely those consisting of maximum concentrations. In addition, the AMP combination treatments revealed parasite strain specificity, such that strains varied in their sensitivity towards the same mixtures. Hence, variable expression of AMPs could be an alternative strategy to combat highly variable infections.This article is part of the themed issue 'Evolutionary ecology of arthropod antimicrobial peptides'.

Metabolic studies of the protozoan parasite, Crithidia luciliae, using proton nuclear magnetic resonance spectroscopy.

Proton nuclear magnetic resonance (NMR) spectroscopy was used to follow glucose metabolism in Crithidia luciliae. Parasites were grown aerobically and anaerobically in culture, with glucose as the major carbon source and 1H NMR spectra were acquired for the cell free medium. The 1H NMR resonances of metabolites utilised and produced during cell growth were identified by difference spectroscopy, and quantitated from standard curves using 3-trimethylsilyl propionate-2,2,3,3-d4 sodium salt as an internal standard. The major metabolites produced by C. luciliae grown aerobically on 8 mM glucose were succinate, pyruvate, acetate and ethanol, in final concentrations in the media when the cells entered stationary phase of 8.5 +/- 0.5, 5.0 +/- 0.3, 2.1 +/- 0.2 and 2.5 +/- 0.6 mM, respectively. The production of succinate and pyruvate, but not acetate and ethanol, followed closely the growth curve of the parasites. Succinate was also measured enzymically and glucose using an autoanalyser. In both cases the results correlated well with the NMR data. The amounts of end products formed were greater than could be accounted for by the utilisation of glucose or any other metabolite observable in the 1H NMR spectra. There was approximately one extra atom of carbon for each molecule of succinate formed, supporting the view that succinate is produced via phosphoenolpyruvate carboxykinase and carbon dioxide fixation. Anaerobically the same major metabolites were produced, but with a decreased ratio of succinate to acetate and ethanol. The formation of glycerol from glucose was not observed under these conditions.

A 31P nuclear magnetic resonance study of Crithidia luciliae.

31P NMR has been used to observe phosphorus-containing compounds in both perchloric acid and KOH extracts and in whole cell suspensions of Crithidia luciliae. Cells were grown in Bone and Steinert medium, or in a modified RPMI culture medium and harvested after about 72 h in mid- to late log phase. 31P NMR spectra of the perchloric acid extracts indicated that 3-phosphoglycerate, which is normally at low concentrations in most cells, was the dominant phosphorus-containing compound in the sugar phosphate region. 3-Phosphoglycerate is the end product of glycosomal glycolysis and our finding is consistent with previous observations of the failure to detect prior glycolytic intermediates. Other metabolites observed were ATP, ADP, NAD(P)+, phosphoenolpyruvate and low molecular weight polyphosphates (PPn, n less than 20). The presence of high-molecular-weight polyphosphates was established by spectra recorded on extracts obtained through subsequent treatment of the insoluble fraction with KOH. 31P NMR experiments on whole cells indicated that the average main internal pH of cells in late-log growth phase was approx. pH 7.2 +/- 0.1, using the orthophosphate resonance as an indicator. The cells responded to the addition of glucose (final concentration approx. 35 mM) with a decrease in pH, both internal (delta pH = -0.9 (55 min)-1) and external (delta pH = -1.3 (15 min)-1). Polyphosphates and ATP could not be observed in whole cell experiments, although perchloric acid extracts of identically treated cells showed no significant depletion of these compounds.

Impaired drug uptake in methotrexate resistant Crithidia fasciculata without changes in dihydrofolate reductase activity or gene amplification.

Crithidia fasciculata cells grown in defined medium are sensitive to methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR). When cells are challenged with 2-5 microM MTX, cell division ceases after 3-4 divisions and the cells become rounded and immotile for approximately 60 h, with a 40% decrease in cell viability occurring during this period. The cells then recover normal morphology and cell division resumes. Cells which undergo this treatment can be transferred directly into high levels of the drug (1-2 mM). The resistance phenotype is stable in the absence of the drug. Resistance correlates with impaired uptake of [3H]MTX, which in wild-type cells is taken up by a carrier-mediated process. There is no indication of gene amplification at the DNA level or at the level of DHFR activity, as occurs in the case of MTX-resistant Leishmania major. Several lines of MTX-resistant L. major which show gene amplification also exhibit impaired uptake of [3H]MTX.

Effects of sulfur-containing analogues of stearic acid on growth and fatty acid biosynthesis in the protozoan Crithidia fasciculata.

A variety of analogues of stearic acid in which one of the methylene groups was replaced by a sulfur atom were examined as inhibitors of growth and fatty acid biosynthesis in the trypanosomatid protozoan Crithidia fasciculata. The 8-, 9-, 10-, and 11-thiastearic acids were found to suppress the synthesis of the cyclopropane-containing fatty acid dihydrosterculic acid (9,10-methyleneoctadecanoic acid) at micromolar concentrations in the growth medium, and all but the 9-thiastearate were found to inhibit the growth of the protozoa at concentrations. The most potent inhibitor, 8-thiastearic acid (I50 for growth = 0.8 microM; I50 dihydrosterculate synthesis = 0.4 microM), was also observed to inhibit the synthesis of gamma-linolenic acid at a similar concentration. The sulfoxide derivatives of the 9- and 10-thiastearates were found to have little effect on growth or fatty acid synthesis, and several long-chain amides of 3-amino-1,2-propanediol were found to have effects similar to those of the fatty acids from which they were derived.

Stimulated transport of adenosine, guanosine and hypoxanthine in Crithidia luciliae: metabolic machinery in which the parasite has a distinct advantage over the host.

Nutritional insufficiency is a common environmental extreme to which parasitic protozoa are routinely exposed. In this study of purine salvage mechanisms we illustrate some successful adaptations of the parasite Crithidia luciliae to its environment, particularly in the case of purine stress. In purine-depleted conditions, the insect trypanosome C. luciliae has the ability to increase the rates of transport of adenosine, guanosine and hypoxanthine and the activity of the exoenzyme 3'nucleotidase (3'NTase) during the growth cycle. The dramatic increase in these activities appears after a 72-h period in culture. The increased activity of the purine transporters and 3'NTase could be suppressed by addition to the medium of a purine supplement such as adenosine or hypoxanthine (100 microM). Under conditions where the concentration of purines in the medium could be closely regulated, C. luciliae grown in purine-replete medium (> or = 75 microM purine) exhibited low rates of purine transport and activity of 3'NTase. In comparison, parasites transferred to medium with a low purine source (< or = 7.5 microM adenosine) had levels of adenosine, guanosine and hypoxanthine transport elevated 25-40-fold. The results link the simultaneous increase in activity of the nucleoside and base transporters, 3'NTase activity and a general increase in the purine salvage of C. luciliae to the concentration of purines available at any time to the parasite.

UV-induced growth inhibition in the trypanosomatid Crithidia fasciculata.

The effects of short ultraviolet (253 nm) radiation on the growth of the trypanosomatid flagellate Crithidia fasciculata were studied in liquid medium and nutrient agar plates. Cell duplication was completely inhibited after exposure of the flagellates to doses equal to or higher than 50 J/m2. The UV-induced lag period was dose-dependent. Survival was reduced to 1% after exposure of the parasites to 200 J/m2. Ultrastructural changes after the lag period were studied by transmission electron microscopy. Changes of the kinetoplast network structure and sometimes of the mitochondrial matrix were observed. The existence of DNA repair mechanisms in this protozoan is discussed.

Auxotrophic mutants of Crithidia fasciculata.

In pursuance of genetic studies, after exposure to ethylmethanesulfonate, 11 auxotrophic mutants of Crithidia fasciculata were cloned. Three proved uracil dependent; 3 serine dependent; and the remainder have not had their auxotrophy defined.

[Effect of quinones and nitrofurans on Trypanosoma mega and Crithidia fasciculata]. / Efecto de quinonas y nitrofuranos sobre Trypanosoma mega y Crithidia fasciculata.

Demonstration of trypanocidal effects in vitro is a first step for the development of new antichagasic drugs. In order to obtain an experimental model allowing the pre-screening of potential trypanocides for Trypanosoma cruzi in a short time and under safe conditions, the trypanosomatids T. mega and C. fasciculata were assayed for their response to a) compounds known for their action on T. cruzi, and b) compounds not tested before on the latter. The drugs were assayed on the organisms growth in a liquid culture medium, cell multiplication being measured by the medium turbidity increase, using a photoelectric colorimeter previously calibrated with cell suspensions of known concentration. A series of quinones (Lapachones and related compounds), naftoquinone-imines, benzoquinones (perezone and dihydroperezone), a quinol (miconidine) and several nitrofurans, including nifurtimox and (5-nitro-2-furfurylidene)-amino (NF-group) derivatives, inhibited the flagellates growth, specially T. mega, with half-maximal inhibitory concentrations lesser than 5.0 microM, for the most active compounds. T. mega response to nifurtimox, NF-derivatives and beta-lapachone was in close agreement with that of T. cruzi. Cultures of T. mega in the presence of NF-pyrazole, NF-indazoles and NF-imidazole but not nifurtimox, showed irreversible damage since, after re-incubation in fresh medium without inhibitor, these cells grew significantly less than their corresponding controls. Similar effects were observed in C. fasciculata, with beta-lapachone and one naftoquinone-imine. Our results qualify T. mega as an adequate experimental model for the assay of antichagasic agents, as C. fasciculata and T. brucei brucei do for the african trypanosomes.

Seasonal variability of prevalence and occurrence of multiple infections shape the population structure of Crithidia bombi, an intestinal parasite of bumblebees (Bombus spp.).

Ergonomic growth phases of annual social insect societies strongly influence horizontally transmitted parasites. Herein, we focused on the impact of temporal changes in host demography on the population structure of a horizontally transmitted parasite. Seasonal fluctuations in prevalence and the occurrence of multiple infections of the gut parasite Crithidia bombi were analyzed in repeatedly sampled populations of two common bumblebee (Bombus spp.) species. Prevalence of C. bombi was greatest in the middle of the foraging season and coincided with the maximal occurrence of multiple infections. Both decline later in the season. The genetic structure of the parasite population also showed strong seasonal fluctuations with a drastic decline in effective population size and an increase in linkage disequilibrium when infection rates were highest. These effects are mainly attributable to significant changes in parasite allele frequencies leading to selection of specific alleles and increasing the frequency of homozygote genotypes in the middle of the season. Within host, competition between parasite genotypes might explain the observed pattern leading to selection of these alleles, and thus a boost of homozygote genotypes in the middle of the season. Toward the end of the season, selection appears to relax and we observed a recovery in linkage equilibrium, as well as an increase in effective population size. This might be explained by genetic exchange in these trypanosomes in natural populations.